Effects of Standard PRP and Extracellular Vesicle-Enriched PRP on Human Gingival Fibroblasts

Study design: This is a laboratory-based study using blood donated by healthy volunteers, with a paired design. Blood from each of six healthy donors will be processed into both standard PRP and EV-enriched PRP, and each donor's PRP will be compared only with the same donor's EV-enriched PRP. Samples will not be pooled; each donor's samples will be analyzed separately. This design aims to minimize the influence of biological variability between donors on the comparison.

Blood collection and product preparation: Two tubes of peripheral venous blood (18 mL in total) will be collected from each volunteer. Standard PRP will be prepared using a commercially available, CE Class IIb certified, single-spin closed-system PRP kit (T-Lab, Turkey; 1150 RCF, 4 minutes). EV-enriched PRP will be prepared using the T-LAB Exomine kit (Turkey), in which Advanced PRX tubes with micro-lyzer blade technology activate platelets and induce EV release through mechanical stress. Stocks of PRP and EV-enriched PRP will be diluted in serum-free DMEM/F12 to final concentrations of 50%, 25%, 10%, 5%, 2.5%, and 1%.

Cell culture: Human gingival fibroblasts (HGF-1, ATCC) will be cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% amphotericin B at 37°C and 5% CO2. Cells at passages 4 to 8 will be used in all experiments.

Cell proliferation assay: HGF-1 cells will be seeded in 96-well plates (2 x 10^3 cells/well) and, after overnight attachment, treated with a negative control (serum-free DMEM), a positive control (DMEM with 10% FBS), six PRP concentrations, and six EV-enriched PRP concentrations (14 groups in total, in technical triplicate). Absorbance will be measured spectrophotometrically at 570 nm at 24, 48, and 72 hours, and relative proliferation (%) will be calculated against the negative control.

Wound healing (scratch) assay: HGF-1 cells will be seeded in 24-well plates (8 x 10^4 cells/well). At 80% confluence a scratch will be created and cells will be treated using the same group layout. Images will be captured with a phase-contrast microscope at baseline, 24 hours, and 48 hours, and the percentage of cell-free area will be calculated with ImageJ software.

Gene expression analysis: After the most effective concentrations for proliferation and migration are identified, HGF-1 cells (5 x 10^4 cells/well, 24-well plates) will be treated with a control (DMEM with 10% FBS) or the optimal PRP and EV-enriched PRP concentrations. On days 3 and 7, expression of TGF-beta1, PDGF-A, PDGF-B, COL1A2, VEGF, and EGF will be analyzed by RT-qPCR, with beta-actin as the reference gene. Relative fold change will be calculated using the 2^-ddCt method.

Sample size: Sample size was based on the large effect sizes reported in the literature for the effects of platelet concentrates on gingival fibroblast function. With a paired t-test, alpha = 0.05, and an assumed effect size of d = 1.5, six donors provide 83% statistical power.

Statistical analysis: Normality will be assessed with the Shapiro-Wilk test. Paired comparisons between PRP and EV-enriched PRP will use the paired t-test or the Wilcoxon signed-rank test, depending on distribution. The combined effects of concentration and time will be evaluated with repeated-measures ANOVA (or the Friedman test) and appropriate post-hoc corrections. p < 0.05 will be considered statistically significant.

Hypothesis: EV-enriched PRP will show superior biological effects on gingival fibroblast proliferation and wound healing compared with conventional PRP.